Method for diagnosing TSE-induced changes in tissues using infrared spectroscopy

ABSTRACT

A method for diagnosing TSE-induced pathologic changes in tissues including the steps of: (a) directing infrared radiation to a tissue sample with pathologic changes caused by TSE, recording its spectral characteristics after irradiation and (b) comparing and classifying the infrared spectra thus obtained with a reference database containing infrared spectra of TSE-infected tissues and non-infected tissues.

A method for diagnosing TSE-induced changes in tissues using infraredspectroscopy.

BACKGROUND OF THE INVENTION

1) Field of the Invention

This invention relates to a method for fast detection of pathologicchanges induced by transmissible spongiform encephalopathies (TSE) inanimal or human tissue using infrared spectroscopy (IR spectroscopy).

2) Brief Description of the Prior Art

Transmissible spongiform encephalopathies are communicableneurodegenerative diseases of the central nervous system (CNS) that mayaffect many mammals and humans. TSE is used as a cover term here thatrefers to the various forms of this disease as they occur in the variousspecies. In addition to scrapie (trotting disease), the disease thatoriginated in sheep but can be transmitted to hamsters and mice, fiveother types of TSE have become known as yet: Bovine spongiformencephalopathy (BSE) in cattle, chronic wasting disease (CWD) in someAmerican deer and elk, transmissible mink encephalopathy (TME) in mink,feline spongiform encephalopathy (FSE) in cats, and a spongiformencephalopathy in antelopes. Four types of TSE are distinguished inhumans: Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinkersyndrome (GSS), fatal familial insomnia (FFI), and kuru.

TSE can definitely be diagnosed based on a) histological proof ofcharacteristic spongiform changes in the brain tissue accompanied bygliosis, b) immunological proof of deposits of the pathologic prionprotein (PrP) using a,Western blot test, histo-blot test, andimmunohistochemistry, c) proof of scrapie-associated (PrP) fibrils (SAF)using an electron microscope, and d) proof of the infectious TSE agentusing transmission experiments in animals.

Clinical symptoms and chemical laboratory findings of increasedconcentrations of specific proteins in cerebrospinal fluid and/or serum[Protein 14-3-3 (Zerr et al. (1997) N. Engl. J. Med. 336: 874; Zerr etal. (1998) Ann. Neurol. 43: 32-40.), Protein S100 (Otto et al. (1997) J.Neurol. 244: 566-570; Otto et al. (1998) Brit. Med. J. 316: S77S82; Ottoet al. (1998) J. Neurovirol. 4: 572-573) as well as neuron-specificenolase (Zerr et al. (1995) Lancet 345: 1609-1610)] in animals andhumans can only give rise to a tentative diagnosis. The same applies tochanges visible in EEGs or MR tomograms that occur in conjunction withhuman TSE.

Development and improvement of detection procedures for TSE serve, interalia, the following purposes.

a) Improving differential diagnostics of human TSEs. These diseases canonly be diagnosed with any certainty by a post mortem or cerebralbiopsy.

b) Detection of TSE contamination in blood, organs, and tissue and inproducts of human or animal origin produced from these.

c) Identification of blood, organ, and tissue donors infected with humanTSE.

d) Detection of preclinical or clinical stages of TSE infection in farmanimals (e.g. cattle and sheep) at the slaughterhouse or farm.

To be able to diagnose TSE diseases in farm animals is important becausethese diseases may be transmitted through eating the meat of diseasedanimals. It is suspected, for example, that the consumption ofBSE-contaminated beef can cause a new variant of CJD in humans (nvCJD).Some states are currently introducing official monitoring ofcontamination levels in cattle populations to protect consumers andcontain the spread of the epidemic. It is envisaged to carry out routinechecks at slaughterhouses to establish whether the carcasses can beused.

Various test systems are being developed to provide sensitive and fastscreening of large sample populations for pathologic prion protein andthus to provide diagnostics for large-scale production. These include acapillary electrophoresis immunoassay using fluorescence-labeledpeptides (Schmerr & Jenny (1998) Electrophoresis 19: 409-419) and animmunological detection system using fluorescent lanthanide chelatescalled Delfia (Safar et, al. (1998) Nature Medicine 4. 1157-1165).

Only one diagnostic method is currently available to identifyTSE-infected farm animals that is suitable for large-scale application.It is restricted to use at slaughterhouses and, according to thedeveloper's statements, can detect BSE in cattle up to half a yearbefore clinical symptoms occur (see the manufacturer's information onthe Internet at http://www.prionics.ch).

In this method developed by Swiss-based Prionics AG, a tissue sampleobtained from the medulla oblongata of slaughtered cattle is homogenizedand treated with proteinase K enzyme. The pathologic prion protein thatmay remain after this treatment is labeled with the 6H4 monoclonalantibody (manufactured by Prionics) and then stained using the Westernblot method. The manufacturer states that it takes up to 12 hours fromtaking the sample to getting a final result using this method. It is theproblem of this invention to develop a method that provides fast,reliable and cost-efficient detection of TSE-induced tissue changes. Themethod according to the invention is to work efficiently in the routineoperations of a slaughterhouse.

It is an object of this invention to provide a method for detectingTSE-induced pathologic changes in tissues. Said changes may be caused byscrapie, BSE, or any other of the TSE diseases. This objective isachieved by the present invention in the following steps: (a) directinginfrared radiation onto a tissue sample and recording the spectralcharacteristics after interaction with the sample; (b) comparing theinfrared spectrum thus obtained against a reference database containinginfrared spectra of TSE-infected tissues and non-infected tissues; and(c) classifying the infrared spectrum as a spectrum obtained fromTSE-infected or non-infected tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart of the method according to the present invention;

FIG. 2 is, in a first example of the present invention, a chart of twotypical spectra of S- and N-tissue samples;

FIG. 3 is, in a second example of the present invention, a dendrogram ofa specific hierarchical spectrum classification as calculated usingWard's-algorithm;

FIG. 4A is, in a third example of the present invention, a dendogramcalculated after data compression using principal component analysis;and

FIG. 4B, in the third example of the present invention, compares normedvector second derivations of sample spectra and differential spectra ofnormed vector S- and N-spectra.

DETAILED DESCRIPTION OF THE INVENTION

The method used in this invention is mainly based upon measurements ofinfrared spectra of pathologically changed tissue. It has been knownfrom a number of publications and patent applications thatdisease-specific changes may be reflected in the infrared spectrum oftissues (U.S. Pat. No. 5,168,162 (Wong & Rigas; U.S. Pat. No. 5,038,039;Wong, Rigas; Lasch & Naumann (1998) Cell. Mol. Biol. 44:189-202; Laschet al. (1998) Proc. SPIE 3257: 187-198; Choo et. al. (1996) Biophys. J.71: 1672-1679). However, no data have been published on infraredspectroscopy performed on TSE tissue samples.

The experimental data this patent description is based on was developedusing CNS samples of scrapie-infected hamsters as a model system. It wasfound in this hamster model that characteristic changes in the infraredspectrum of CNS tissue samples occurred after the animals were infectedwith scrapie. These changes were identified using the method accordingto the invention, i.e. by comparing respective samples obtained fromnon-infected healthy animals. In principle, this method can be used forthe diagnosis of any clinical picture of the TSE group of diseases.According to the method of the invention, TSE diagnosis using infraredspectrography requires comparing spectra of the tissue to be examinedwith spectra of tissues of known origin for reference. Practicalapplication of this method therefore requires the existence of avalidated reference database of IR spectra obtained from healthy andpathologic tissue samples. This reference database has to be createdjust once to perform a standardized diagnostic method.

Matching spectra obtained from unknown samples with the referencedatabase can be performed using methods of computeraided patternrecognition such as multivariate statistics, artificial neuronalnetworks, genetic algorithms, etc.

The spectra are obtained by directing infrared light onto the samplesand recording the spectral characteristics of the emerging radiation,i.e. after the light has interacted with the tissue. Use ofmicrospectrometric techniques is advantageous when minimization ofsample sizes is desired. When an infrared microscope is used, spectraldata can be obtained from thin slices of tissue in positionalresolution, which makes the method considerably more specific andsensitive. In future improvements, an infrared optical waveguide couldbe used as an endoscope and facilitate TSE diagnosis directly in theinfected organism.

A typical flowchart of the method is shown in FIG. 1. The new method ofdetecting TSE-induced changes in tissue enables users to make statementswithin one minute from obtaining the sample. This makes it superior toimmunological detection of the prion protein and immunohistologicaldiagnosis, as these provide results after up to 12 hours only. Sovirtually no intermediate storage of the carcasses is required to waitfor test results when the method according to the invention is usedduring routine operation. Fastness of this diagnostic method representsan economic advantage as compared to known methods because it minimizesthe storage time of the carcasses and thus space and energy costsrequired for refrigerating them. In addition, the meat is fresher at thetime of final consumption.

This method can easily be integrated into a routine process as the stepsof recording, processing, and classifying the spectrum are fullycomputer-controlled and easily automated. Just a few staff members arerequired for the relatively simple process of sample preparation, butunlike other methods, pretreatment of the samples does not require amajor effort (such as enrichment of the prion protein by proteinase Kdigestion) or staining thin slices of tissue (by immunohistologicalmethods).

The method according to the invention can be performed without involvinghighly specialized professionals (such as histologists) as the IRspectra are classified using generally known methods of computer-aidedpattern recognition that are optimized for the purposes of TSEdiagnosis. As the spectra are evaluated according to strict mathematicalcriteria, diagnosis is highly reliable, does not require inference fromexperience, thus bypassing human misjudgment.

The method of the invention has an economically reasonable design due toits moderate staffing requirements and virtually no material costsduring operation.

The advantage of the method according to the invention in its specificIR microscopy embodiment for analyzing thin slices of tissue inpositional resolution is that it combines structural information inspectral resolution with the high positional resolution obtained fromit. Involvement of individual neurons in the pathogenesis can berecorded and studied using the mapping an IR microscope can provide. Thevery high diagnostic sensitivity of the method results from the factthat characteristics of diseased and healthy cells are practically notaveraged, which is bound to happen with methods that do not offermapping functionality. This specific embodiment of the method stillrequired relatively much time for data collection, which may take from 1to 6 hours depending on the size of the tissue area under review and thepositional resolution. However, it should be widely used in scientificand clinical studies of the pathogenic mechanisms of TSE which have notyet been understood. In the future, this embodiment will be combinedwith so-called array infrared detectors that are being developed byvarious manufacturers and will be capable of measuring IR spectra ofcomplete thin slice areas in positional resolution and in a very shorttime, making this method eventually suitable for fast routine diagnosis.

The method of the invention first requires taking post mortem tissuesamples from the organism. Samples may be taken from animal and humanorganisms.

The method is suitable for detecting each of the special clinical formsthat are covered by the term ‘transmissible spongiform encephalopathy’(TSE), such as BSE, scrapie, or CJD.

All organs that show TSE-induced pathologic changes can be used as sitesfor collecting tissue samples. As far as we know today, affected organsare the central nervous system, the peripheral nervous system, organs ofthe lymphatic system, the digestive system, the endocrine system, thecardiovascular system, and the respiratory system. Preferred collectionsites are the central nervous system and the peripheral nervous system,the medulla oblongata and the Varolian pons being particularlyadvantageous. The tissue sample is prepared depending on the specificway the method is carried out.

Small pieces of tissue are collected for analyzing fully hydrated tissuesamples. The native samples are placed into commercial IR cuvets.Alternatively, a homogenizate of the tissue material in H₂O is produced,and aliquots are placed in IR cuvets. In a variation of the method,aliquots of this suspension are dried up as transparent films onIR-transmitting sample holders; the drying process is accelerated byreduced pressure (Helm et al. (1991) J. Gen. Microbiol. 137:69-79).

Cryostat sections are made for carrying out the method in an IRmicroscopy measuring arrangement for collecting specific locally mappeddata. These are evenly applied to IR transparent microscopic slides. Themethod does not require any fixing of the thin slice of tissue. Thesamples are stored at a dry place at room temperature until they aremeasured.

In another embodiment that uses infrared waveguides, the method can beapplied in vivo by introducing the waveguide into the tissue usingminimal invasion technique and directly collecting the infrared spectrumfrom there. This embodiment requires an improvement of infraredwaveguide technology to become practicable as currently availablewaveguides still have a too low spectral sensitivity, are too inflexibleand too big. Materials suitable for cuvets or sample holders/slides forthe preparation variants described above are all water-insoluble opticalmaterials conventionally used in IR spectroscopy, while CaF₂ and BaF₂have proven particularly useful.

The amount of substance required for IR spectra and their superficialextent can be very small. Depending on the conditions set (such asspectroscopy with or without beam focusing or using an IR microscope),sample sizes in the μg to ng ranges can be used. The diameters of theirradiated sample areas vary between 1-3 mm and 10-30 μm. The lowerlimit is about the size of one or a few cells (e.g. neurons).

According to the method of the invention, the infrared spectra of tissuesamples that were prepared in the way described are measured. Thespectra are preferably taken using a Fourier transform infraredspectrometer which has a number of known advantages as compared toconventional disperse equipment, including fast data collection andhigher sensitivity. It would generally be possible to use a conventionaldisperse IR spectrometer but this would slow the method down. Inprinciple, each of the generally known IR spectrometry setups (such astransmission/absorption, diminished total, direct, or diffusereflection) can be used to measure the spectra. Transmission/absorptionspectroscopy has proven particularly useful.

The infrared spectrum is typically taken in the midinfrared spectralregion, i.e. between 500 and 4000 cm⁻¹. Narrower spectral regions evenin near infrared range from 4000 to 10000 cm⁻¹ can also providesuccessful diagnosis if the user made sure that the spectra of infectedand healthy tissue samples show characteristic variance in the spectralregion recorded. It was found, in particular, that marked spectraldifferences between TSE-infected and non-infected tissues were detectedin the range from 1000 to 1300 cm⁻¹, and that this range is particularlysuited for diagnosis.

One or several suitable spectral regions can be selected by visualinspection of spectra (selecting the ranges that show the strongest andmost characteristic changes as compared to the control group) or by agenerally known multivariate method for selecting spectralcharacteristics.

The physical parameters such as spectral resolution or number ofaveraged spectra, etc. can be varied within the typical ranges in IRspectroscopy without having any critical practical influence on thesuccess of classification or diagnosis. When determining the parametersfor obtaining the spectra and preparing the sample, identical parametershave to be selected for all measurements including control measurementsof tissue samples from non-infected animals.

Conditioning of the spectra has proven advantageous no matter whichmathematical-statistical method was chosen for spectrum classification.The generally known methods that can be used here include calculation ofthe first or second derivation, deconvolution, or other methods toincrease spectral contrast, to facilitate band recognition, and tominimize any baseline problems that may occur. When sample populationsare large, upfront data reduction using methods of multivariatestatistics such as factor analysis has proven helpful.

The method requires one-time creation of a database of referencespectra. Spectra of samples from TSE-infected organisms and fromnon-infected individuals are measured. Samples are prepared and spectrataken in the same way as with unknown samples. It is important that allparameters for reference and sample measurements are identical.

The spectrum of the sample to be examined is compared with the spectrastored in the reference database. The spectrum is preferably classifiedusing a method of pattern recognition such as algorithms of multivariatestatistics, artificial neuronal networks, or genetic algorithms. Thisstep classifies a spectrum based on a two-class problem as being eitherhealthy or TSE-infected.

When the method is carried out in positional resolution, the sample (athin slice of tissue applied to a microscopic slide) is placed in thebeam path of an infrared microscope. The spectra can be taken bytransmission or reflection in the infrared spectrometric arrangement.Infrared spectra are taken from various tissue areas. Positionalresolution can be determined by the increment between measuringpositions. It is very advantageous to use a computer-controlled X-Ystage that facilitates automated spectral measurements according to afreely selectable grid with defined increments. Such X-Y stages arestandard accessories of state-of-the-art IR microscopes.

The result of a measurement in position resolution (mapping) is a seriesof infrared spectra wherein each spectrum represents a pixel on afictitious grid of the thin slice of tissue. In this way, IR data isobtained that completely covers the selected area of the thin slice oftissue. The result of a measurement in position resolution (mapping) isa series of infrared spectra wherein each spectrum represents a pixel ona fictitious grid of the thin slice of tissue. In this way, IR data isobtained that completely covers the selected area of the thin slice oftissue. Position-specific information about the expansion of TSE in thetissue is maintained by comparing each mapping record with the referencedatabase and thus classifying it as being healthy or infected. Thefollowing examples are to illustrate the way in which CNS samplesobtained from scrapie-infected hamsters can be distinguished fromsamples obtained from healthy control animals based on thedisease-specific modifications in their infrared spectra using themethod according to the invention.

EXAMPLE 1

Adult female Syrian hamsters (Mesocricetus auratus) were intracerebrallyand intraperitoneally infected with the 263K scrapie strain (provided byDr. Richard Kimberlin). In the terminal stage of the disease (70 to 120days after infection), the brains of these animals (S) and of matchingnoninfected control animals were removed post mortem; correspondingpairs for comparison were of similar age. Small pieces (μg-scaleamounts) of the natively resected medulla oblongata and Varolian ponswere put into an FTIR cuvet equipped with CaF₂ windows and an opticalpath length of 8 μm (thickness of layer). The infrared spectra of thesesamples were measured in transmission/absorption using an FTIRspectrometer (spectral resolution: 4 cm⁻¹, apodization: Happ-Genzel,number of scans: 126, zero filling: 4). Two typical spectra of S- andN-tissue samples are shown in FIG. 2 in the spectral region from 1300 to1000 cm⁻¹ in which differences that can be observed are particularlyprominent. The second derivations are represented for improvedvisualization of the bands, showing the band peaks as minima.

EXAMPLE 2

In a variation of the embodiment described in Example 1, 10 S- and 10N-samples obtained as in Example 1 were homogenized in H₂O (10 μl of H₂Oper mg of tissue material). Aliquots of 35 μl of the suspensions wereapplied to a PC-controlled multiple sample holder made of ZnSe that isalso suitable for measuring microbial samples (Helm et al. (1991) J.Gen. Microbiol. 137:69-79; Heim et al. (1991) J. Microbiol. Meth.14:127-142; Neumann (1998) Proc. SPIE 3257:245-257), and dried asdescribed in the literature. The infrared transmission spectra of thefilms thus obtained were taken and classified hierarchically based onsecond derivations of these spectra in the spectral region from 1100 to1000 cm⁻¹. FIG. 3 shows the dendrogram of this spectrum classificationas calculated using Ward's algorithm. The spectra of infected animals(S-3 through S-10) could be perfectly distinguished from those of thehealthy animals (N-1 through N-10 ).

EXAMPLE 3

In a variation of the embodiments described in Examples 1 and 2,cryostat sections were created of CNS samples obtained from N- andS-animals as described above, and measured and characterized using thegenerally known method of FTIR mapping (Diem et al. (1999) Appl.Spectroscopy 53: 148A-160A; Lasch & Neumann (1998) Cell. Mol. Biol. 44;189-202; Choo et al. (1996) Biophys. J. 71:1672-1679) and infraredimaging (Lasch & Neumann (1998) Cell. Mol. Biol. 44:189-202; Lasch etal. (1998) Proc. SPIE 3257:187-198). Spectra were taken of 1.5 mm by 1.5mm areas in increments of 50 μm through an aperture of 60 μm. Thespectra obtained from the S- and N-samples were first separatelyclassified in hierarchies to differentiate the typical spectra for thevarious brain structures. FIG. 4A shows a dendrogram that was calculatedafter data compression using principal component analysis based on thefirst three principal components between 1450 and 950 cm⁻¹ (ca. 500 datapoints). The four main classes can be assigned to the fourhistologically defined cerebral structures: molecular layer, ganglioncell layer, granular cell layer, and white substance matter. Inaddition, nine spectral classes (numbered 1 to 9) were separated thatcorrespond to specific structures within the cerebellum. FIG. 4A onlyshows each third spectrum of the mapping record that contains 930spectra for better clarity. Subsequently, the spectra of correspondingspectral classes (e.g. class 2 of the molecular layer spectra—the graysubstance of the cerebellum) of the N- and S-samples were compared. Theupper part of FIG. 4B (a) compares normed vector second derivations ofsample spectra from scrapie-infected animals (dashed line) and fromhealthy animals (solid line). The lower part shows differential spectraof normed vector S- and N-spectra from a) for the respective tissuestructures. All spectra used for this comparison are averages of spectraof a spectral class A, FIG. 4A). They are identified by the name oftheir cerebellar layer and the number of their spectral class. Thecharacteristic spectral differences observed for each tissue class aresuitable for reliably diagnosing the disease-associated pathogeneticprocess.

We claim:
 1. A method for diagnosing transmissible spongiformencenhalopathies-induced (TSE-induced) pathologic changes in tissues,said changes being caused by scrapie, bovine spongiform enceghalopathies(BSE, or another disease of the TSE group of diseases, comprising thesteps of: (a) directing infrared radiation onto a tissue sample andrecording the spectral characteristics after interaction with thesample; (b) comparing the infrared spectrum thus obtained against areference database that contains infrared spectra of TSE-infected andnon-infected tissues; and (c) classifying the infrared spectrum as aspectrum obtained from TSE-infected or non-infected tissues.
 2. Themethod according to claim 1, wherein said tissue sample is collectedfrom one of the central nervous system, the peripheral nervous systemand human organs.
 3. The method according to claim 2, wherein the humanorgans are from one of the lymphatic system, the digestive system, theendocrine system, the cardiovascular system and the respiratory system.4. The method according to claim 2, wherein said infrared spectrum ofthe tissue is measured in at least one region of one of the mid-infraredrange from 500 to 4000 cm⁻¹ and the near infrared range from 4000 to10000 cm⁻¹.
 5. The method according to claim 2, wherein said infraredspectrum of the tissue is measured in the spectral region from 1000 to1300 cm⁻¹ of the mid-infrared range.
 6. The method according to claim 1,wherein said infrared spectrum of the tissue is measured in at least oneregion of one of the mid-infrared range from 500 to 4000 cm⁻¹ and thenear infrared range from 4000 to 10000 cm⁻¹.
 7. The method according toclaim 2, wherein said infrared radiation interacts with said sample, andthe characteristically altered radiation is detected in one of atransmission/absorption, attenuated total reflection, direct reflectionmeasuring setup, diffuse reflection measuring setup, and by using IRwaveguides.
 8. The method according to claim 6, wherein said infraredradiation interacts with said sample, and the characteristically alteredradiation is detected in one of a transmission/absorption, attenuatedtotal reflection, direct reflection measuring setup, diffuse reflectionmeasuring setup, and by using IR waveguides.
 9. The method according toclaim 1, wherein said infrared spectrum of the tissue is measured in thespectral region from 1000 to 1300 cm⁻¹ of the mid-infrared range. 10.The method according to claim 1, wherein said infrared radiationinteracts with said sample, and the characteristically altered radiationis detected in one of a transmission/absorption, attenuated totalreflection, direct reflection measuring setup, diffuse reflectionmeasuring setup, and by using IR waveguides.
 11. The method according toclaim 1, wherein said infrared spectrum of the sample to be examined iscompared against the reference database using at least one method ofpattern recognition, and that the spectral regions said comparison isbased on are determined using methods for extracting optimum spectralcharacteristics.
 12. The method according to claim 11, wherein the atleast one pattern recognition method uses algorithms of one ofmultivariate statistics and artificial neuronal networks.
 13. The methodaccording to claim 11, wherein the extracting optimum spectralcharacteristic method uses genetic algorithms.
 14. The method accordingto claim 1, wherein said infrared spectrum is measured on a thin sliceof tissue using an IR microscope set up for one of transmission anddirect reflection spectrometry.
 15. The method according to claim 14,wherein said infrared spectra are measured in positional resolution. 16.The method according to claim 14, wherein each mapped infrared spectrumis compared against the reference database, thereby providing localizedinformation on the spread of the disease in the tissue.
 17. The methodaccording to claim 14, wherein said reference database containsreference spectra of TSE-infected tissues and non-infected tissues ofall structures that can be distinguished in the tissue section usinginfrared spectroscopy.
 18. The method according to claim 14, whereinsaid infrared spectra are mapped to the tissue site where the infraredbeam is transmitted through the sample.
 19. The method according toclaim 18, wherein each mapped infrared spectrum is compared against thereference database, thereby providing localized information on thespread of the disease in the tissue.